Nanocapsules having a polyelectrolyte envelope

ABSTRACT

A method for the production of nano- or microcapsules having a diameter of from 20 nm to 40 μm is suggested, wherein template particles are supplied in an aqueous medium, electrically recharged with a polyelectrolyte, re-recharged without separation or washing steps using a second polyelectrolyte having a complementary charge with respect to the first polyelectrolyte, and said process is optionally continued with alternately charged polyelectrolytes.

[0001] The invention relates to nanocapsules enclosed by a stable coatlayer of polyelectrolytes, to a method of producing such structures, andto the use of these structures. The invention also relates to a devicefor the production of said nanocapsules.

[0002] Liposomes are known to be a highly biocompatible form ofpackaging various active substances. The components of liposomes aretolerable at high doses, triggering no or only slight defensivereactions by the immune system. However, the use of liposomes frequentlyis opposed by their sensitivity to mechanical, thermal or biologicalexposure.

[0003] The lifetime of liposomes can be extended via the composition ofthe liposomal membrane; however, this is accompanied by loss of otherdesirable properties such as fusion competence.

[0004] Up to now, numerous efforts have therefore been made to utilizethe mild and biocompatible way of packaging by liposomes with the aid ofstabilizing additives for uses in pharmacy and industry. A familiarmethod of increasing the stability of liposomes is doping their surfacewith various polymers, particularly polyethylene glycol (PEG). Thesecomponents effect steric shielding of the surface, thus preventingdirect attack of lytic components, e.g. from the blood system, on themembrane; e.g. “stealth liposomes” as liposomal preparations wherein theliposomes are enclosed by a coat of PEG (D. D. Lasic, “Liposomes—fromphysics to applications”).

[0005] Other well-known methods use protection of the liposomal membraneby coating sugar oligomers on the membrane layer. Similarly, stericshielding of the membrane surface is achieved by the coated components.In contrast to non-modified liposomes, the structures obtained can befrozen or lyophilized.

[0006] DE 198 52 928.7 and WO 00/28972 disclose coat structures onliposomal templates, which structures allow versatile modification, theyare stable by themselves and can be produced using layer-by-layerchemisorption of polymers or bio-molecules. Apart from producing coatlayers and nanocapsules, the method also allows for biocompatiblemodification and functionalization of the surface.

[0007] Alternatively, structures of similar constitution can be producedusing layer-by-layer polyelectrolyte self-assembly on colloidaltemplates (Caruso, F. (1998), Science 282, 1111-1113, DE 198 12 083 A1,EP 0,972,563 A1, and WO 99/47253)

[0008] WO 00/03797 discloses the suitability of liposomes and otherbiological templates as supports for the production of nanocapsulesusing layer-by-layer self-assembly.

[0009] A familiar method of producing such structures is crosslinking ofproteins on boundary surfaces (U.S. Pat. No. 5,498,421) or on thesurface of liposomes (Kupcu, S., Sara, M., and Sleytr, U. B., Biochem.Biophys. Acta, 1235 (2), 263-269 (1995)).

[0010] U.S. Pat. No. 5,308,701 discloses a method which describesinclusion of liposomes, among other things, in microcapsules made up ofpolyelectrolyte layers. However, the solution described therein avoidsbinding of the first polyelectrolyte to the lipid layer. Rather, theliposomes in U.S. Pat. No. 5,308,701—like all the other dissolvedsubstances—are micro-encapsulated by a droplet of the surroundingpolymerizing material. The liposomes do not act as a template of themicro-capsule; the result is formation of significantly larger capsulesincluding a multitude of liposomes in their gel-like interior. As aresult of their size, these micro-capsules are unsuitable for use in theblood circulation.

[0011] Furthermore, the well-known basic structures involve thefollowing drawbacks:

[0012] U.S. Pat. No. 5,498,421 uses an oil phase as matrix which, as aconsequence, allows inclusion of fat-soluble substances only. As aresult, use of this system for most biopolymers is impossible.

[0013] Being highly immunogenic structures, the S-layer proteins used byKupcu et al. are unsuitable for use in pharmaceutical carriers.

[0014] Furthermore, the disclosed liposomal structures and methods ofproducing same are disadvantageous due to lacking bio-compatibility andin that dissolution thereof involves extreme conditions such as hightemperatures or exceedingly low pH values.

[0015] Moreover, the well-known methods use excess polyelectrolytematerial to a achieve a preferably dense and reproducible coating on thesurface of the nanocapsules or liposomes and therefore, additionalprocess steps are required to remove excess polyelectrolyte material. Inaddition, there is no disclosure of a method for coating liposomes withpolyelectrolytes. The methods proposed in general, e.g. in WO 00/03797,are not feasible due to formation of undissolvable aggregates during theprocess. Another drawback is that all these processes proceed in adiscontinuous fashion, preventing uniform coating of the nanocapsules orliposomes.

[0016] It was therefore the object of the invention to provide aneconomic method allowing facile production of templates, especiallyliposomes enclosed by an independent coat layer.

[0017] The present invention solves this technical problem by providingnano- or microcapsules having a diameter of between 20 nm and 40 μm,template particles being supplied in an aqueous medium, electricallyrecharged with a first polyelectrolyte, re-recharged without separationor washing steps using polyelectrolytes of complementary charge, saidprocess optionally being continued with alternately chargedpolyelectrolytes. The alternating charge of the layers can be generatedin such a way that e.g. the first layer is comprised of anionic orpredominantly anionic polyelectrolytes and the second layer of cationicor predominantly cationic polyelectrolytes.

[0018] Templates in the meaning of the invention are all those templateswhich can be coated using the method according to the invention.Subsequent to providing the templates, the particles can be coated e.g.in an amphiphilic fashion and coated in a further step with apolyelectrolyte which, in particular, has a charge opposite to that ofthe surface of the particle materials. In order to form multiple layers,the templates are subsequently treated with polyelectrolytes of oppositecharge, i.e. alternately with cationic and anionic polyelectrolytes. Thepolymer layers undergo self-assembly on the previously charged solidtemplates by electrostatic deposition in layers, thereby forming amulti-layered polymer coat around the solid cores.

[0019] According to the invention, such structures of alternatelycharged polyelectrolytes can be produced e.g. by coating two or morewater-soluble polyelectrolyte layers of complementary charge onto thesurface of liposomes which, in particular, are capable of producingelectrostatic interactions. The polymer electrolyte layers in directsuccession have opposite charges. Any number, but at least two suchpolymer electrolyte layers can be deposited on the surface of theliposomes. Polyelectrolytes in the meaning of the invention are polymerswith groups capable of ionic dissociation, which groups can becomponents or substituents of the polymer chain, their number being of alevel so as to render the polymers water-soluble in their dissociatedform. According to the invention, ionomers are concerned in that casewhere the concentration of ionic groups is insufficient for watersolubility. Polymers having not more than one or only a few ionic groupsare macroions, e.g. macroanions or macrocations. In the meaning of theinvention, the polyelectrolytes are subdivided in polyacids andpolybases, depending on the type of dissociable groups. Polyanions whichcan be both inorganic species and organic polymers are formed frompolyacids upon dissociation with abstraction of protons. Examples ofpolyacids—the salts of which being referred to as polysalts—are thefollowing: polyphosphoric acid, polyvinylsulfuric acid,polyvinylsulfonic acid, polyvinylphosphonic acid, and polyacrylic acid.Polybases include such groups as pro-ionic groups which, inter alia, arecapable of accepting protons e.g. by reaction with acids to form salts.Typical polybases with chain or lateral dissociable groups arepolyethyleneimine, polyvinylamine and polyvinylpyridine.

[0020] In the meaning of the invention, polyelectrolytes including bothanionic and cationic groups as substituents on a macromolecule arepolyampholytes.

[0021] Polyelectrolytes dissociate into polyions and the correspondingcounterions. As a rule, they are readily soluble in the aqueous solutionof their counterions. In solution, their macromolecules mostly have alinear orientation as a result of electrostatic repulsion between theionic groups, while in non-dissociated form they are present as coiledmolecules. Oligo- and polyvalent counterions induce cross-linking of thepolyelectrolytes which may go up to a level resulting in insolubilitythereof.

[0022] According to the invention, the polyelectrolytes can bebiopolymers such as alginic acid, gum arabic, nucleic acids, pectins,proteins, etc., as well as chemically modified biopolymers, e.g.carboxymethylcellulose, ligninsulfonates and synthetic polymers, e.g.poly(meth)acrylic acid, polyvinylsulfonic acid, polyvinylphosphonicacid, polyethyleneimine. Other polyelectrolytes are well-known to thoseskilled in the art, e.g. from WO 00/28972 or WO 00/03797, the contentsthereof being incorporated in the disclosure of the invention.

[0023] For example, both structure-forming and activity-bearing polymerscan be used to build up the polyelectrolyte layer. The coats may havebinding properties for other molecules or catalytic properties, forexample. The polymers with structure-forming and activity-bearingproperties can be found e.g. among proteins. According to the invention,it is possible to utilize e.g. hemoglobin to build up the coatstructure. Thus, the nanocapsules of the invention can be used as bloodsubstitute. However, it is also possible to integrate proteins in thepolyelectrolyte layer which are capable of recognizing and bindingcharacteristic structures of other proteins. In particular, suitableproteins for this purpose are lectins as well as proteins binding biotinor antibodies. Such nanocapsules are capable of recognizingglycosylations, antigenic epitopes or biotin groups on proteins or othermacromolecules, binding these components in a highly specific manner.

[0024] As starting material, liposomes or template particles can beused, the size of which determining the size of the nanocapsules beingformed. Suitable methods of producing such liposomes are well-known tothose skilled in the art. According to the invention, the liposomes usedmust enable binding of the water-soluble polymer, in particular. Methodsof covalent coupling in aqueous media are well to those skilled in theart and involve, among other things, heterofunctional and homofunctionallinkage of amino, thiol, hydrazo, hydroxo, acidic hydrogen, aldehyde,and carboxyl groups, or activated esters thereof in suitablecombinations.

[0025] As a result of the characteristics of the lipid layer, theinteraction between the liposome and the first polyelectrolyte maydiffer from the preferred electrostatic interactions between succeedinglayers. One possible embodiment of the teaching according to theinvention therefore involves using such amphipatic polymers orpolyelectrolytes which, in combination with a lipid layer, yield acharged particle. Examples of such polyelectrolytes are integral ormembrane-bound proteins, amphipatic polymers such as alkyl acrylates,alkyl-modified sugar polymers, and other such substances of natural orsynthetic or semi-synthetic origin.

[0026] In a succeeding step, a second layer including another polymer iscoated on the template particle coated with the first polymer. Thecharges of the first and second polymer are complementary to each other.First and second polymer form a network on the surface of the liposomes.

[0027] The polymers used have a zeta potential other than zero under thereaction conditions. Important quantities to bias this potentialcomprise the pH value of the solution and the ionic strength. Suitablecompounds include a variety of polyelectrolytes, as well as otherwater-soluble polymers with sufficiently polar groups. For example,suitable compounds include: polysaccharides such as alginic acid,chitosan, pectin, hyaluronic acid, polymannuronic acid, polygalacturonicacid, heparin, gum arabic, Indian traganth, xanthan gum, Carragheen,locust bean gum, and the salts of these compounds, as well ascarboxylated, aminated, hydrazylated dextrans, starches, levans,inulins, or agaroses.

[0028] Other compounds are natural or synthetic proteins or peptides orother homo- or heteropolymers of amino acids, oligonucleotides, DNA orRNA in single-stranded or double-stranded form and in linear orcircular-closed form, synthetic polymers such as polyacrylic acids,polyacrylamides, polyacrylic esters, and other polymers of derivativesof acrylic acid, polyvinylpyrrolidones, polyethyleneimines,polystyrenesulfonic acids, polyallylamines, polyphosphazenes, etc.Further compounds are hetero- or block polymers of the above basicmonomers. Also included are mixed forms of the above-mentionedcompounds, such as glycosylated proteins, proteins modified subsequentto translation, protein complexes with other natural materials,complexes of proteins and nucleic acids, copolymers of sugars andacrylates and related compounds, as far as all of these compounds are tobe water-soluble, in particular.

[0029] Following two or more coatings, polyelectrolyte-coatednanocapsules are obtained wherein, in particular, a lipid membrane isenclosed by an exterior coat. Advantageously, this coat alters thesurface properties of the liposomes, increasing the stability thereof.The stability can be increased further by exposure to chemicalcrosslinkers, for example.

[0030] In particular, the coating reaction according to the method ofthe invention can be performed quite rapidly and therefore, chemicalcrosslinkers advantageously can be mixed into the suspension as early asat the beginning of the reaction and conveniently removed from thesuspension not before completion of the coating reaction, if essentialfor subsequent use.

[0031] In particular, the method allows completion of the coatingreaction within a few seconds, said reaction rarely taking longer than afew minutes.

[0032] Suitable crosslinkers are all those compounds specified e.g. inWO 00/28972 and, in particular, those crosslinkers having no or nosubstantial effect on the electric net charge of the polyelectrolytes.

[0033] In particular, the liposomes employed must allow binding of thefirst water-soluble polymer. Suitable components for producing suchliposomes are charged amphipatic compounds which can be incorporated inthe lipid layer without destroying same, which is most important.Suitable compounds include natural or synthetic phospholipids andderivatives thereof, particularly phosphatidyl serine, phosphatidylinositol, phosphatidyl glycerol, or phosphatidic acid, as well assphingolipids, ceramides, tetraether lipids or other ether lipids, aswell as derivatives of cholesterol like cholesterol sulfate, cholesterolhemisuccinate, dimethylaminoethylcarbamoylcholesterol and other suchcompounds. Suitable compounds also include alkylcarboxylic acids,alkylsulfonic acids, alkylamines, alkylammonium salts, dialkylamines orammonium compounds like DOTAP or DOTIM, phosphoric esters withlong-chain alcohols and other membrane-forming or membrane-boundcompounds. Non-charged membrane components such as phosphatidyl choline,phosphatidyl ethanolamine, α-tocopherol, cholesterol etc. can also beused as membrane-forming components.

[0034] In particular, both the liposomes and the polymers used have aplurality of charges which ultimately can lead to binding of thecomponents to each other and to the nanocapsules of the invention.

[0035] The liposomes may have uni- or multilamellar membrane structures.It is preferred to use uni- or oligolamellar liposomes having a size ofbetween 20 and 1000 nm, preferably between 50 and 500 nm, and morepreferably between 70 and 300 nm.

[0036] As a result of the rapid succession of individual mixingprocesses, the aggregation tendency of the mixtures can be reduced withadvantage, thereby enabling coating even at higher lipid and polymerconcentrations; in particular, preservation of the integrity of theliposomes in the continuous coating process is superior compared to thediscontinuous stirring process, for example.

[0037] In a preferred embodiment, the mixing chamber is a staticmicromixer resulting in particularly rapid and uniform mixing, even withsmall flows of liquid. Suitable mixers have been described in DE 199 25184 A1.

[0038] This variant of the method works largely independently of thenature of liposome or template used in coating. The benefits of rapid,flow-optimized production of nanocapsules without any intermediate stepcan also be utilized with colloidal liposomes or templates described sofar. Advantageously, the method can be used in the production ofpolyelectrolyte coats on non-stable templates. Thus, for example, it isalso possible to stabilize droplets of an oil-in-water emulsion by usingthis method,

[0039] In another preferred embodiment of the invention, the liposomesare dissolved subsequent to coating with polyelectrolytes, preferably byleaching with a detergent. Thus, structures in the form of hollowspheres may be formed, wherein the liposomes have been dissolvedsubsequent to crosslinking. This may result in liberation of polymersthat are bound to the lipid layer only, and not to each other, and maygive rise to decomposition of insufficiently crosslinked structures. Thenanocapsules can be separated from the decomposition products bysedimentation, gel filtration or ultrafiltration.

[0040] Suitable detergents to dissolve the interior liposomes arealkylated sugars such as octylglucoside, salts of cholic acid andderivatives thereof, alkylsulfonic acids, polyoxyethylenesorbitol, orsimilar compounds. The nanometer range nanocapsules in the meaning ofthe invention therefore are merely comprised of a polymer backboneforming the surface of a sphere. In particular, the shaping liposomescan be removed in such a way that the size of the hollow spheres havingformed is determined by the liposomes being used.

[0041] Advantageously, the permeability of the coat layer of thenanocapsules can be increased substantially by leaching the liposomes.For example, this process involves the passage of detergent moleculesand mixed micelles through the outer coat layer. In the same way,substrates and products of a reaction proceeding in the interior of thehollow sphere can be exchanged. One arrangement for performing suchreactions preferably consists of hollow spheres having in their interiorenzymatically active substances with high molecular weights, theliposomes of which have been leached by detergents. In particular,suitable substances for such an inclusion are enzymes or ribozymes.However, it is also possible to leach non-bound polymers only, so thatthe lipid layer is preserved. In that case, only those substancesdiffusing through the lipid layer can be exchanged. These areamphiphilic molecules such as phenylalanine. In particular, nanocapsulesincluding phenylalanine-4-hydroxylase or phenylalanine ammonia-lyase canbe used to decompose particular amino acids in phenylketonuria.

[0042] In another preferred embodiment of the invention, the coat layerof polyelectrolytes is subjected to covalent crosslinking withbifunctional reagents subsequent to deposition thereof.

[0043] In another advantageous variant of the method, natural orsynthetic polymers or mixed forms of these compounds, e.g. polyacids orpolybases are used as polyelectrolytes. Inter alia, polyacids are formedin the dissociation of polyanions, and these polyanions can be bothinorganic and organic polymers. Polybases include groups which, inparticular, are capable of accepting protons.

[0044] In another embodiment of the invention, the polyelectrolytescomprise alginic acids, chitosan, nucleic acids, polynucleotides and/orproteins, preferably albumin, hemoglobin, myoglobin, antibodies,proteases, α₂-macroglobulin, fibronectin, collagen, vitronectin, proteinA, protein G, avidin, streptavidin, concanavalin A, and/or wheat germagglutinin.

[0045] In another variant of the method, active substances are to beenclosed in the nanocapsules. For example, active substances can bebiologically or chemically active compounds which, at lowconcentrations, have a qualitative or quantitative effect on chemical,biochemical, biophysical, and physiological processes, e.g. metabolicprocesses in living organisms in a way so as to activate or inhibitparticular processes. For example, active substances occurring naturallyin organisms, such as vitamins or hormones, can be used. However, it isalso possible to use exogenic active substances such as biocides.

[0046] In another variant of the method, the liposomes are to comprisephosphatidyl serine, phosphatidyl glycerol, phosphatidic acid,sphingolipids, ceramides, tetraether lipids, cholesterol sulfate,cholesterol hemisuccinate, dimethylaminoethylcarbamoylcholesterol,alkylcarboxylic acids, alkylsulfonic acids, alkylamines, alkylammoniumsalts, dialkylamines, DOTAP, DOTIM, phosphoric esters with long-chainalcohols, phosphatidyl choline, phosphatidyl ethanolamine, and/orα-tocopherol.

[0047] In another embodiment of the invention, coating with polymers isperformed at a lipid concentration lower than 2 mM. It is preferred touse lipid concentrations lower than 1 mM, more preferably lower than 0.5mM, and most preferably lower than 0.2 mM. Owing to the dilutionsselected, it is possible in an advantageous fashion to suppressformation of aggregates.

[0048] In another embodiment, liposomes including 10 to 50 mole-%,preferably 30 to 50 mole-%, and especially 35 to 45 mole-% of chargedsterols are to be used in the method.

[0049] When using phospholipids, it is convenient to use more than 10mole-%, preferably more than 40 mole-%, and more preferably more than 60mole-%.

[0050] Advantageously, the amount of deposited polymer and thus, thedensity of the generated layers can be controlled via the density ofcharge carriers on the liposome. This finding is surprising in that thecharge carriers can be mobile within the liposomal membrane, andrelatively low amounts are sufficient for stoichiometric saturation ofthe polymer. If the charge carriers themselves are membrane-formingsubstances, e.g. charged phospholipids or derivatives thereof, orcharged dialkyls like DOTAP or DOTIM, even higher amounts of chargecarriers can be used with advantage. In this event, it is preferred touse amounts of between 10 and 100% of total lipid, more preferablyamounts between 40 and 100%, and most preferably amounts between 40 and80% of the above-mentioned substances. Advantageously, the chargecarrier density on the surface can be increased further by using groupswith multiple charges, e.g. high amounts of phosphatidic acid with twonegative charges, or substituted membrane-bound compounds bearingmultiple charges, e.g. conjugates of spermine and sterols, or those ofoligopeptides and phospholipids, or those of heparin and lipids, orthose of other multifunctional compounds such as oligo- andpolycarboxylic acids or oligo- or polyamines and lipids. The transitionto lipid/polymer conjugates is fluid, and the examples mentioned can besupplemented easily by a person skilled in the art.

[0051] In another embodiment of the invention, coating with polymers isperformed at a salt concentration of more than 50 mM.

[0052] Advantageously, the density of liposomal charge carriers can bedetermined via the maximum salt concentration of the solution wherecomplete binding of the polymer to the template still takes place.Performing coating at a salt concentration as high as possible isadvantageous for two reasons:

[0053] (i) Salt concentrations of more than 50 mM result in significantcompacting of highly charged polymers because intramolecular repulsionis reduced, thereby enabling denser packing of polyelectrolytes on thesurface.

[0054] (ii) Though not invariably, but in many cases, subsequentincrease of the salt concentration of the medium results in aggregationof the particles, probably by partial destabilization of the outerpolyelectrolyte layer. However, reducing the salt concentration does notdo damage.

[0055] In particular, the coating reaction—even in thedilutions—requires considerably less time than that to be expectedaccording to the prior art. Such rapid progression of the reactionenables purposeful arrangement of a continuous process.

[0056] Surprisingly, it has also been determined in this context thatthe phase transition temperature of the liposomal membrane has an effecton the reaction rate. Thus, the coating reactions proceed more rapidlywhen coating the lipid membrane above the phase transition temperature.

[0057] In a preferred embodiment of the invention, one reaction cycle isto be completed within less than 20 minutes, preferably within less thanfive minutes, and more preferably within less than one minute.

[0058] In another advantageous embodiment of the invention, a chemicalcrosslinker is added during the coating reaction or subsequent tocompletion thereof.

[0059] In another preferred embodiment of the invention, the templateparticles are to have a size of between 20 nm and 1000 nm, preferablybetween 50 nm and 500 nm, and more preferably between 70 nm and 300 nm.

[0060] In another preferred embodiment of the invention, two or moredissimilar polyelectrolytes are coated simultaneously or successively inone layer.

[0061] In a preferred embodiment, the template particles are liposomes.

[0062] It may be convenient to have the template particles in anoil-in-water emulsion. In particular, it may be convenient if theemulsions include active substances in their oil phase.

[0063] In a preferred embodiment of the invention, the nanocapsulesadditionally have a lipid layer which has the polyelectrolyte layersthereon. For example, the lipid layer can be the outer oil layer ofliposomes situated in the nanocapsule.

[0064] The invention also relates to structures including lipid layersin the interior thereof; a liquid phase may be present in the interiorof said structures. In principle, any liquid, also including suspensionsin the meaning of the invention, can be contained in the interior of thenanocapsules. Examples of liquids are water, buffers, fluid aerosols,etc.

[0065] It may be convenient if the structures include a water-immiscibleoil phase in the interior thereof.

[0066] In another embodiment of the invention, the structures includeactive substances. Active substances in the meaning of the invention aresubstances which—occurring or added in relatively small amounts—arecapable of developing physiological effects. Examples of such activesubstances are hormones, vitamins, enzymes, trace elements,pharmaceutical agents, feed additives, fertilizers, pesticides, etc.

[0067] Inter alia, the active substances in the meaning of the inventioncan have qualitative or quantitative effects, be it activation orinhibition, on biochemical and physiological processes in livingorganisms.

[0068] In another preferred embodiment of the invention, the activesubstances are part of the polyelectrolyte layer or lipid layer of saidstructures. Advantageously, it is possible in this way to incorporatelipid-soluble components, e.g. fat-soluble vitamins, at highconcentrations in the nanocapsules.

[0069] In another preferred embodiment of the invention, the activesubstance is a catalyst, a biocatalyst, a pharmaceutical agent, anenzyme, a pharmaceutical substance, a protein, a peptide, anoligonucleotide, a sensor, nucleic acids, and/or a crystal. For example,the active substances can be entrapped in the nanocapsules, or, ifliposomes are present in the nanocapsules, the above-mentioned activesubstances can also be entrapped in the liposomes. In this case,liposomes can be used which already include the substances to beentrapped. Methods of producing such liposomes are well-known to thoseskilled in the art. Substances which can be used are specified in thatthey must not adversely affect the integrity of the liposomes, as wouldbe the case with detergents. Suitable substances are e.g. proteins,peptides, vitamins, hormones, carbohydrates, or nucleic acids, as wellas mixtures thereof. Suitable substances also include antibiotics,fungicides and antiviral agents, cytostatic agents and immunosuppressiveagents, analgetic agents, anesthetics, antidepressive agents,antidiabetic agents, antihypertensive agents, anticoagulants,antiinflammatory agents, anxiolytic, sedative, antiarrhythmic,antiarthritic active substances, bronchodilators, hypoglycemic andhypolipidemic active substances, as well as active substances tostimulate erythropoiesis and apoptosis-inducing substances. For theinclusion of cargo molecules, it is also possible to start withliposomes already including these substances or having these substancesbound thereto. The entrapped or bound substances remain in the interiorliposomes or in the lipid layer during all of the reaction steps.

[0070] The invention also relates to the use of the inventivenanocapsules as containers or vehicles in pharmaceutical formulations.

[0071] In particular, the coated liposomes are used as containers andvehicles for biologically active substances.

[0072] As a result of the variety of usable components, the coatedliposomes and the nanocapsules free of lipids can be used in numerousapplications. The use of the nanocapsules expands the spectrum ofcarrier materials in the sense of drug targeting, as a transfer vector,a sustained release form, or in an enzyme substitution therapy.Advantageously, the components being used can be both structure-formingand activity-bearing. In particular, the coated liposomes and thenanocapsules free of lipids can be produced using substances having anantigenic effect or substances which do not induce an immune response.

[0073] The use of the nanocapsules is made possible as a result of theadvantages of the method according to the invention which, for the firsttime, combines the advantages of mild inclusion of active substances inliposomes with an efficient technology of coating colloidal particles,which can be implemented without using further auxiliary agents andtherefore involves particular advantages in pharmaceutical uses.

[0074] Enzymatic or fluorescent properties of the nanocapsules areadvantageous for use in detection systems. Suitable substances havingsuch properties are green fluorescent protein or phycobiliproteins.Other suitable polymers can be modified using fluorescent substances.Per se, suitable methods are known to those skilled in the art andinvolve covalent binding of the activated fluorophore to appropriategroups in the polymer, or complex formation of fluorescent metal ionswith chelating groups of the polymer.

[0075] Amongst proteins, there are polymers with enzymatic activity,such as peroxidases, phophatases, proteases, dehydrogenases,glucosidases, etc.

[0076] However, nanocapsules having such a structure can also be used intarget-controlled application of drugs. These highly specific moleculestherefore include particularly those capable of interacting with thesurface of cells. Complementary pairs in this sense are antibodies andmembrane-bound antigens, lectins or selectins, and membrane-boundglycosylations, hormones and receptors thereof, and others. The modulardesign of these structures is advantageous, allowing the generation of afree number of specificities on just a few coat layers on the one hand,and a highly economic use of the components ultimately determining thespecificity, on the other hand. The valency of the structure that isobtained, i.e., the number of surface-bound components determining thespecificity, can easily be modified by titration. A high density ofthese components is equivalent to high avidity, enabling stableinteractions even in case of unfavorable binding constants of eachsingle interaction, as is the case e.g. between MHC complexes and T-cellreceptors.

[0077] In another advantageous embodiment of the invention, nanocapsulesonce formed are modified with other substances. One important variant ofthis embodiment is modification of the nanocapsule surface usingpolyethylene glycol or sugars or other polyalcohols. Such coatingresults in particles having improved compatibility in pharmaceuticaluses. When using the structure described herein for entrapping enzymes,the architecture open to diffusion ensures high availability of theentrapped activity. In addition, the diffusion paths are extremely shortin the selected micrometer and submicrometer size ranges. Other uses arein the production of microcrystals of specific sizes on a chemical orbiochemical route.

[0078] In another use, particularly those enzymatically activesubstances are employed whose substrates and products can be exchangedthrough the coat layer.

[0079] Nanocapsules in the meaning of the present invention have astructure open to diffusion, allowing exchange of molecules ofsignificant size, e.g. during dissolving of the lipid layer. However,large molecules such as enzymes can be retained by the coat layer. Inother inventive uses of the nanocapsules, they are loaded with enzymescatalyzing reactions, the substrates and products of which are capableof passing through the coat layer. Compared to the prior art, this wayof enveloping a biological macromolecule in nanocapsules offers theadvantage of extremely short diffusion paths and an associated increaseof the specific activity of the entrapped enzyme. In addition, exposureto crosslinking agents as encountered in chemical fixation can beavoided.

[0080] However, signal-generating systems such as horseradish peroxidaseor alkaline phosphatase or fluorescence-labelled macromolecules havingspecific binding properties for other substances can also be entrappedin such nanocapsules. Such systems are suitable in the detection of saidother substances, particularly in medical or biochemical diagnostics.Compared to liposomes, an advantageous fact is that nanocapsules arestable to detergents, particularly those detergents used to suppressnon-specific binding in such procedures, such as Tween 20 or TritonX-100.

[0081] In one variant of this use according to the invention, thenanocapsules themselves are the carriers of the signal-generatingsystem. Advantageously, nanocapsules are prepared wherein the polymershave fluorescent properties. To this end, fluorescent derivatives of P1and/or P2 are used to build up the nanocapsules, or the nanocapsules arecoupled covalently to fluorescent substances subsequent to theirpreparation.

[0082] In one inventive use of the nanocapsules, they are designed so asto specifically bind to target cells of mammals. Nanocapsules used inthis sense have one or more classes of ligands on their surface, thecomplementary binding counterparts of which being situated on thesurface of the target cells. Nanocapsules having such properties arevehicles of therapeutic agents, directing the latter to a well-definedsite of action. In such a use, the inner lipid layer of the hollowspheres can be maintained if beneficial in entrapping the substance tobe transported.

[0083] In one variant of this use according to the invention, thenanocapsules include substances against which an immune response is tobe triggered.

[0084] In one advantageous variant of this embodiment of the invention,the nanocapsules are used to transfer active substances into the cytosolof mammal cells. These nanocapsules are designed so as to beincorporated by mammal cells via endocytosis. Nanocapsules used in thisembodiment of the invention are comprised of a coat layer which can bedigested by the hydrolases of the endosome. Moreover, they are producedusing liposomes whose membrane is capable of fusing with that of theendocytotic vesicle. One advantage in this embodiment of the teachingaccording to the invention is represented by the fact that such a fusioncannot give rise to liberation of lytic endosomal activities into theinterior of the cell. Nanocapsules for this purpose can be loaded withvarious active substances. However, the above-described path oftransport is particularly advantageous in transporting biologicalmacromolecules incapable of membrane permeation, such as proteins,peptides, antibodies, enzymes, oligonucleotides, DNA, RNA, hormones, butalso antibiotics, fungicides and antiviral agents, as well as cytostaticagents.

[0085] In another advantageous embodiment of the invention, thenanocapsules are to be used in biochemical diagnostics.

[0086] In another advantageous embodiment of the invention, thenanocapsules are to be used in the production of microcrystals,herbicides, pesticides and/or pigments. For example, microcrystals inthe meaning of the invention are materials consisting of one or moresubstances and having a microscopic order. For example, peptidemolecules can be concerned. Herbicides in the meaning of the inventionare substances capable of effecting a negative modification in thedevelopment of all wild and cultivated plants that are undesirable intheir respective location. For example, these can be defoliants,herb-destroying agents or other substances present in the form of anaerosol, liquid or solid. The herbicides in the nanocapsules of theinvention can be used in pre-seeding, pre-emergence and post-emergenceof cultivated plants. The respective active substances can be selectedin such a way that the nanocapsules according to the present applicationare incorporated in the soil or applied in the foliage area of theplant. In particular, contact herbicides are concerned in that casewhere the herbicides develop their effect directly at the site ofcontact. Specifically, the following can be used: inhibitors ofphotosynthesis, respiration, growth substances, germination, carotenesynthesis, and others. Pesticides in the meaning of the invention areall formulations capable of neutralizing or destroying all harmful oroffensive organisms or prevent exposure thereto. For example, these mayinclude agents against flies, horseflies, mosquitoes, cockroaches,bedbugs, or fleas etc., as well as products to be used against rats,mice, beetles, or moths. Pigments in the meaning of the invention areessentially insoluble, inorganic or organic, colored or non-coloreddyes.

[0087] The invention also relates to a device for the continuous coatingof liposomes in multiple polyelectrolyte layers, wherein a mixer isprovided in the main flow of the liposomes effected by a pump,downstream of an attenuator for each separately supplied inflow ofrequired polyelectrolyte effected by a pump, one timing element at atime being arranged between the mixers, and one attenuator at a timebeing arranged between the pumps for polyelectrolyte feeding and themixers.

[0088] Accordingly, the teaching of the invention incorporates anapparatus for the continuous coating of liposomes with multiplepolyelectrolyte coats. A pump allows for constant flow of the liposomesolution within a tube system. The polyelectrolytes used for coating areintroduced successively into the tube system as shown in FIG. 1. Mixingof the reactants is effected by strong flow or using dynamic or staticmixers in the feed points. The volume of the tube sections between theindividual mixing points is dimensioned such that, at a given flow rate,sufficient reaction time is available until the next mixing point isreached.

[0089] In a special embodiment, the device can be of such a design thatthe tube line constitutes the timing element.

[0090] The nanocapsules according to the present invention involveseveral advantages; they are hydrophilic, permeable and detergent-stablestructures of crosslinked polymers, which, as a result of the variety ofusable components, can be specified for a large number of applications.The present invention considerably expands the spectrum of substanceswhich can be used as carrier materials in the sense of drug targeting,as a transfer vector, a sustained release form, or in an enzymesubstitution therapy. The components being used can be bothstructure-forming and activity-bearing. The hollow spheres described canbe produced using substances having an antigenic effect or substanceswhich do not induce an immune response.

[0091] Surprisingly, it has been found that the method according to thepresent application, which has many advantages compared to the priorart, allows for virtually complete binding of the employed polymer tothe liposomes when using suitable mass ratios, so that separation stepsbetween single coatings are no longer required. This fact cruciallycontributes to the process economy of the method The suitable quantityof layer material required each time roughly corresponds to the maximumamount bound under the given reaction conditions and can be derivedtherefrom.

[0092] To determine the suitable amount of polyelectrolyte for eachparticle, increasing amounts of polymer are titrated to a suppliedsuspension of particles in small batches, and the size of the particlesis determined thereafter. The size of the liposomes rapidly increases byformation of aggregates with the polymer to exceed a maximum. When thesize with increasing amounts of polymer comes back to its originalvalue, the optimum amount suitable for coating is used.

[0093] Surprisingly, a number of polymers, particularly proteins, werefound to have a low tendency of forming aggregates. Using thesematerials, particles of any coating stage, i.e., template particles orspecies already coated can be subjected to an initial coating in such away that recharging does not yet occur. Recharging then is achieved byaddition of the same, or an equally charged, yet materially differentpolyelectrolyte. In this way, individual layers can be doped somewhatwith activity-bearing molecules. The well-dosed use of specificallybinding components permits variation of the binding strength of theparticle.

[0094] One important variant of the teaching of the invention is thatliposomes are contacted with polyelectrolytes of complementary charge asearly as during the process of their formation. Such liposomes compriseentrapped polyelectrolyte molecules, as well as polyelectrolytemolecules adhering to the outside. The aggregation tendency decreaseswith the concentration of polyelectrolyte, and it is preferred to useless than 500 μg/mg lipid, more preferably less than 150 μg protein/mglipid.

[0095] In dilute suspensions according to the above-described preferredconcentrations, such particles are stable for from several minutes tohours and can be coated further according to the method of theinvention. It is an advantageous effect that separation or washing stepsneither are required when entrapping active substances.

[0096] Surprisingly, it has also been found that the amount of depositedpolymer and thus, the density of the generated layers can be controlledvia the density of charge carriers on the liposome.

[0097] Another advantage over the prior art is that the undesirableprocess of aggregate formation can be suppressed by high-speed mixing ofthe reactants and by using suitable dilutions. Another advantage is thatthe density of the liposomal charge carriers can be determined via themaximum salt concentration of the solution.

[0098] In familiar processes, massive formation of aggregates occursduring the production of such mixtures, resulting in flocculation of thereactants. Advantageously, this undesirable process can be suppressed byhigh-speed mixing of the reactants, by using suitable dilutions, and bythe immediate succession of the individual steps.

[0099] Without intending to be limiting, the invention will be explainedin more detail with reference to the following examples.

EXAMPLES Abbreviations

[0100] CTAB Cetyltrimethylammonium bromide FITC Fluoresceinisothiocyanate PC Phosphatidyl choline MES2-(N-Morpholino)ethanesulfonic acid PSS Polystyrenesulfonic acid PEIPolyethyleneimine DPPC Dipalmitoylphosphatidyl choline DPPGDipalmitoylphosphatidyl glycerol DOPE Dioleoylphosphatidyl ethanolamineCHEMS Cholesterol hemisuccinate HEPESN-(2-Hydroxyethyl)piperazine-N′-(2- ethanesulfonic acid PLLPoly-L-lysine PAS Polyacrylic acid BSA Bovine serum albumin

Example 1 Nanocapsules Made of Polystyrenesulfonic Acid andPolyethyleneimine

[0101] Preparation of the Liposomes

[0102] 400 mg of PC from soy and 9.7 mg of CTAB are dissolved in ethanoland evaporated to dryness under vacuum. The lipid film is subsequentlyrehydrated using a buffer (10 mM MES, 150 mM NaCl, pH 6.5). Thereafter,the suspension is pressed repeatedly through isoporous polycarbonatemembranes having a pore size of 0.2 μm.

[0103] Coating with PSS

[0104] PSS (Mr 70,000) is dissolved in MES buffer (10 mM, pH 6.5) at aconcentration of 10 μg/ml. The liposomes are diluted with the samebuffer so as to make a lipid concentration of 200 μg/ml. Equal volumesof the two solutions are combined with stirring. Subsequently, thesuspension is concentrated using tangential dialysis.

[0105] Coating with PEI

[0106] PEI (Mr 60,000) is dissolved in MES buffer (10 mM, pH 6.5) at aconcentration of 5 μg/ml. The PSS-coated liposomes are diluted with thesame buffer so as to make a lipid concentration of 200 μg/ml. Equalvolumes of the two solutions are combined with stirring. Subsequently,the suspension is concentrated using tangential dialysis.

[0107] Further coatings can be coated as in the two steps above. Theamount of polymer used binds with sufficient completeness to theparticle surface, so that no purification steps have to be effected inbetween.

Example 2 Analysis of the Structures Having Formed

[0108] The intensity of the scattered light generated by a particlesuspension is measured in a dynamic light scattering apparatus.Following addition of detergent, the measured intensity with liposomesdrops to less than 5% of the initial value. After coating with three ormore polymer layers, more than 40% of the intensity remains.

[0109] The stability of the thus-obtained hollow spheres free ofliposomes is tested by adding NaCl. The particles are stable at least to1 M NaCl.

Example 3 Stability of the Structures in Serum

[0110] That coated liposomes of Example 1 are concentrated byultrafiltration to make a lipid concentration of 1 mg/ml andsubsequently mixed with an equal amount of human serum. The intensity ofthe light scattered by the particle suspension is measured in a dynamiclight scattering apparatus. At the same time, the size of the particlesis determined. 24 hours after addition of serum, more than 90% of theparticles having their initial size can be detected.

Example 4 Preparation of Fluorescent Nanocapsules

[0111] Modification of PEI

[0112] 100 mg of PEI is dissolved in 10 ml of borate buffer (0.1 M, pH9.0) and added with 1 ml of fluorescein isothiocyanate (10 mg/ml indimethylformamide). The mixture is incubated at room temperatureovernight. Fluorescent PEI is purified using gel filtration on SephadexG-25®. A buffer comprised of 10 mM MES and 150 mM NaCl, pH 6.5, is usedto elute the column. The eluted PEI can be detected via its scatteredlight in a dynamic light scattering apparatus, and the fluorescein labelis detected using the absorption thereof. Fractions including a constantratio of fluorescein and PEI are combined and used in subsequentcoating.

[0113] The liposomes are prepared as in Example 1.

[0114] Coating with PSS is effected as in Example 1.

[0115] Coating with Modified PEI

[0116] Fluorescein-labelled PEI is dissolved in MES buffer (10 mM, pH6.5) at a concentration of 10 μg/ml. The PSS-coated liposomes arediluted in the same buffer so as to make a lipid concentration of 200μg/ml. Equal volumes of the two solutions are combined with stirring.Subsequently, the suspension is concentrated using tangential dialysis.

[0117] Further coatings can be coated as in the two steps above. Theamount of polymer used binds with sufficient completeness to theparticle surface, so that no purification steps have to be effected inbetween.

Example 5 Inclusion of a Fluorescent Cargo

[0118] Preparation of the Liposomes

[0119] 400 mg of PC from soy and 9.7 mg of CTAB are dissolved in ethanoland evaporated to dryness under vacuum. The lipid film is subsequentlyrehydrated using a carboxyfluorescein solution (100 mMcarboxyfluorescein, 10 mM MES, 150 mM NaCl, pH 6.5). Thereafter, thesuspension is pressed repeatedly through isoporous polycarbonatemembranes having a pore size of 0.2 μm. Non-entrapped carboxyfluoresceinis removed by gel filtration on Sephadex® G25.

[0120] Coating

[0121] The liposomes are coated with PSS and PEI as in Example 1. Atotal of five layers is coated, the outer and inner layers consisting ofPSS.

Example 6 Coating with PLL as a Function of Liposomal Charge Density

[0122] Preparation of the Liposomes

[0123] 10-100 mole-% DPPG and supplementing amounts of DPPC aredissolved in isopropanol and evaporated to dryness under vacuum. Thelipid film is subsequently rehydrated using an amount of buffer (10 mMHEPES, 150 mM NaCl, pH 7.5) so as to make a lipid concentration of 25mM. The suspension is frozen at least once, thawed at 50° C. andsubsequently pressed repeatedly through isoporous polycarbonatemembranes having a pore size of 200 nm.

[0124] Coating with PLL and Analyzing the Structures

[0125] PLL (70-150 kDa) is dissolved in buffer (10 mM HEPES, pH 7.5) ata concentration of 1 mg/ml. Various liposomal formulations are dilutedin buffer (10 mM HEPES, pH 7.5) to a lipid concentration of 0.2 mM. From0 to 250 μg PLL/mg lipid is supplied in a volume of 0.2 ml at maximumand added with 10 ml of liposomal formulation with agitation.Thereafter, the structures having formed are measured using dynamiclight scattering (see FIG. 2). The size of the liposomes rapidlyincreases by formation of aggregates with the polymer to exceed amaximum. When the size with increasing amounts of polymer comes back toits original value, the optimum amount suitable for coating is used.

Example 7 Coating with PLL as a Function of Salt Concentration

[0126] Preparation of the Liposomes

[0127] As in Example 6, and as a supplement, liposomes are also preparedusing 0 . . . 40% CHEMS and supplementing amounts of DPPC.

[0128] Coating with PLL and Analyzing the Structures Formed

[0129] PLL is dissolved in buffer (10 mM HEPES pH 7.5) at suitableconcentrations (0-230 μg/ml). The liposomal formulations are diluted inbuffer (10 mM HEPES, pH 7.5) to a lipid concentration of 0.2 mM. Sodiumchloride solutions of from 0 to 5 M in buffer (10 mm HEPES, pH 7.5) areprepared. A polymer/salt matrix is built up in 96-well microtiter platesusing 30 μl of the various PLL or NaCl solutions each time. Each well ofa plate is added with 240 μl of a liposomal formulation, and theturbidity is measured at 405 nm after 10 minutes.

[0130] The following table specifies the suitable amount of polymer (μgPLL/mg lipid) required to generate stable, non-aggregated structures.Liposomal Salt concentration charge carrier 10 mM 25 mM 50 mM 75 mM 100mM 150 mM 300 mM 10% DPPG 25 100 Aggr. Aggr. Aggr. Aggr. Aggr. 33% DPPG60 100 130 130 150 250 Aggr. 66% DPPG 130 150 150 150 150 170 Aggr. 100%DPPG 220 230 230 230 250 270 >300 10% CHEMS 25 Aggr. Aggr. Aggr. Aggr.Aggr. Aggr. 20% CHEMS 25 70 Aggr. Aggr. Aggr. Aggr. Aggr. 30% CHEMS 6070 100 250 Aggr. Aggr. Aggr. 40% CHEMS 70 110 115 140 240 Aggr. Aggr.

[0131] Salt Stability of PLL-Coated Liposomes

[0132] Liposomes coated at a particular salt concentration with asuitable amount of PLL, so as to obtain stable structures, are unstablewith increasing salt concentration, forming aggregates.

Example 8 Nanocapsules Made of Albumin and Heparin

[0133] Preparation of the Liposomes

[0134] 20 mole-% DPPC and 80 mole-% DPPG are dissolved in isopropanoland evaporated to dryness under vacuum. The lipid film is subsequentlyrehydrated using an amount of buffer (10 mM HEPES, 150 mM NaCl, pH 7.5)so as to make a lipid concentration of 25 mM. The suspension is frozenat least once, thawed at 50° C. and subsequently pressed repeatedlythrough isoporous polycarbonate membranes having a pore size of 200 nm.

[0135] Coating with Albumin and Heparin and Analyzing the Structures

[0136] The polymers are dissolved in buffer (10 mM sodium acetate, pH 4)at concentrations of 1 mg/ml and 5 mg/ml. The liposomes are diluted inbuffer (10 mM sodium acetate, pH 4) to a lipid concentration of 0.2 mM.50 ml of the diluted liposomes are mixed successively with suitableamounts of the two polymers (see Table). The amounts of polymer usedeach time bind with sufficient completeness to the particle surface, sothat no purification steps have to be effected in between. Layer (S) mgpolymer/mg lipid S1 BSA 1.00 S2 Heparin 0.33 S3 BSA 4.75 S4 Heparin 1.59S5 BSA 12.66 S6 Heparin 4.22

[0137] Crosslinking of the Structures Having Formed UsingGlutaraldehyde, and Concentrating

[0138] For crosslinking, the structures obtained are added withglutaraldehyde. The reaction proceeds over 2 hours at 37° C. and at afinal concentration of 0.15% glutaraldehyde. Thereafter, the solution isadjusted to pH 7.5 using 1 M NaOH. Subsequently, the suspension isdialyzed against 100 mM NaCl using tangential dialysis and thenconcentrated.

[0139] Salt Stability of the Crosslinked Structures

[0140] The stability of the structures obtained is tested by addingNaCl. The particles are stable at least to 150 mM NaCl.

Example 9 Nanocapsules Made of PLL and PAS

[0141] Preparation of the Liposomes

[0142] 60 mole-% DOPE and 40 mole-% CHEMS are dissolved inisopro-panol/chloroform (3:1) and evaporated to dryness under vacuum.The lipid film is subsequently rehydrated using an amount of buffer (10mM HEPES, 150 mM NaCl, pH 7.5) so as to make a lipid concentration of 25mM. The suspension is frozen at least once, thawed at RT andsubsequently pressed repeatedly through isoporous polycarbonatemembranes having a pore size of 200 nm.

[0143] Coating With PLL and PAS and Analyzing the Structures

[0144] PLL (Mr 70 . . . 150 kDa) and PAS (Mr 30 kDa) are dissolved inbuffer (10 mM HEPES, 100 mM NaCl, pH 7.5) at concentrations of 1 mg/mland 5 mg/ml. The liposomes are diluted in buffer (10 mM HEPES, 100 mMNaCl, pH 7.5) to a lipid concentration of 0.2 mM. To produce the firstlayer, 130 μg PLL/mg lipid is supplied in a volume of 1 ml at maximumand 50 ml of the liposomes are injected. To produce the second layer, 55μg PAS/mg lipid is supplied and the PLL-coated liposomes are injected.An analogous procedure is used for the following layers (see Table). Theamounts of polymer used each time bind with sufficient completeness tothe particle surface, so that no purification steps have to be effectedin between. Layer (S) μg polymer/mg lipid S1 PLL 130 S2 PAS  55 S3 PLL200 S4 PAS 200 S5 PLL 850 S6 PAS 800

[0145] Crosslinking of the Structures Having Formed Using EDC, andConcentrating

[0146] For crosslinking, the structures having formed are added withEDC. The reaction proceeds over 12 hours at RT and at a finalconcentration of 50 mM EDC. Thereafter, the crosslinking reaction isquenched using potassium acetate (final concentration 100 mM).Subsequently, the suspension is diatangential using tangential dialysisand then concentrated.

[0147] Salt Stabiliity of the Crosslinked Structures

[0148] The stability of the structures obtained is tested by addingNaCl. The particles are stable at least to 150 mM NaCl.

Example 10 Tolerability of the Structures in Pharmaceutical Uses

[0149] Liposomes with chemically crosslinked polyelectrolyte coats areproduced as in Example 8 or 9.

[0150] Wistar rats (male, 250 . . . 300 g) are kept using a regularday-night rhythm and feeding ad libitum. Two animals at a time areanesthetized, receiving 500 μl of particle suspension via the tail vein.The animals are observed for various periods of time and subsequentlydecapitated and disscted.

[0151] For injection, the following was used specifically: Example 8, S4and S5, and Example 9, S6.

[0152] All of the treated animals survived the injection for at least 24hours. None of the animals exhibited a behavior deviating from normal.Likewise, no lesions in the organs were detected.

Example 11 Coating of an Emulsion

[0153] 2 g of olive oil, 8.5 g of water, 120 mg of phosphatidyl choline,and 250 mg of glycerol are mixed and stirred for two hours.Subsequently, the emulsion is homogenized in an ultrasonic bath andextruded once through a polycarbonate filter having a pore width of 200nm. An emulsion with a mean particle size of 315 nm is formed.

[0154] PLL (70-150 kDa) is dissolved in buffer (10 mM HEPES, pH 7.5) ata concentration of 1 mg/ml.

[0155] 40 μl of emulsion is diluted in 10 ml buffer (10 mM HEPES, pH7.5). From 0 to 50 μg of PLL is supplied and added with 1 ml of emulsionwith agitation. Thereafter, the structures having formed are measuredusing dynamic light scattering. The size of the particles rapidlyincreases by formation of aggregates with the polymer to exceed amaximum. When the size with increasing amounts of polymer comes back toits original value, the optimum amount suitable for coating is used.Suitable amounts of other polyelectrolytes for subsequent build-up oflayers are likewise determined using this method.

[0156] With reference to the drawing, the device of the invention in oneembodiment will be illustrated in more detail below. The drawingrepresents the basic structure of the device in a block diagram.

[0157] The main flow section of the nano- or template particles to becoated is formed by a pump 10 effecting said main flow, an attenuator 20arranged downstream of said pump, and mixers 30, 31, 32, 33, 34, 3X, onetiming element 42, 43, 44, 45, 4X at a time being arranged between saidmixers.

[0158] Each influx section for feeding the respective polyelectrolytesA, B, C, D, E, X is formed by pumps 11, 12, 13, 14, 15, 1X, andattenuators 21, 22, 23, 24, 25 and 2X, respectively, arranged betweenthe pumps and mixers 30, 31, 32, 33, 34, and 3X, respectively.

[0159] The respective modules (pumps, attenuators, mixers, and timingelements) are connected in their arrangement by means of tube lines. Thevolumes of the respective tube lines between mixers 30 and 31, 31 and32, 32 and 33, 33 and 34, 34 and 3X, respectively, form the timingelements 41, 42, 43, 44, 45, and 4X, respectively. The device can beexpanded at will by lining up additional influx sections (represented bydotted lines in the drawing).

[0160] To coat liposomes with multiple polyelectrolyte coats, theliposome solution is passed through the device at constant flow using apump. The polyelectrolytes used in coating are introduced successivelyinto the system of the main flow section. Mixing of the reactants iseffected by means of mixers 30, 31, 32, 33, 34, 3X at the feedingpoints. The volume of the tube lines between each single mixer 30 to 3Xis dimensioned such that, at a given flow rate, sufficient reaction timeis available until the next mixer is reached.

1. A method for the production of nano- or microcapsules having adiameter of from 20 nm to 40 μm, characterized in that templateparticles are supplied in an aqueous medium, electrically recharged witha polyelectrolyte, re-recharged without separation or washing stepsusing a second polyelectrolyte having a complementary charge withrespect to the first polyelectrolyte, said process optionally beingcontinued with alternately charged polyelectrolytes.
 2. The methodaccording to claim 1, characterized in that one reaction cycle iscompleted within less than 20 minutes, preferably within less than 5minutes, and more preferably within less than one minute.
 3. The methodaccording to claim 1 or 2, characterized in that a chemical crosslinkeris added during a coating reaction or subsequent to completion thereof.4. The method according to any of the preceding claims, characterized inthat the template particles have a size of between 20 nm and 1000 nm,preferably between 50 nm and 500 nm, and more preferably between 70 nmand 300 nm.
 5. The method according to any of the preceding claims,characterized in that two or more dissimilar polyelectrolytes are coatedsimultaneously or successively in one layer.
 6. The method according toany of the preceding claims, characterized in that a salt concentrationof more than 50 mM in an aqueous solution is used.
 7. The methodaccording to any of the preceding claims, characterized in that thetemplate particles are liposomes.
 8. The method according to any of thepreceding claims, characterized in that the liposomes are dissolvedsubsequent to coating, preferably using a detergent.
 9. The methodaccording to any of the preceding claims, characterized in that theliposomes have active substances entrapped therein.
 10. The methodaccording to any of the preceding claims, characterized in thatphosphatidyl serine, phosphatidyl glycerol, phosphatidyl inositol,phosphatidic acid, sphingolipids, ceramides, tetraether lipids,cholesterol sulfate, cholesterol hemisuccinate,dimethylaminoethylcarbamoylcholesterol, alkylcarboxylic acids,alkylsulfonic acids, alkylamines, alkylammonium salts, dialkylamines,N-[1-(2,3-dioleoyloxypropyl ]-N,N,N-trimethylammonium chloride,phosphoric esters with long-chain alcohols, phosphatidyl choline,phosphatidyl ethanolamine, and/or α-tocopherol are used in theproduction of the liposomes.
 11. The method according to any of thepreceding claims, characterized in that coating with polymers isperformed at a lipid concentration lower than 2 mM, preferably lowerthan 1 mM, more preferably lower than 0.5 mM, and most preferably lowerthan 0.2 mM.
 12. The method according to any of the preceding claims,characterized in that liposomes including 10 to 50 mole-%, preferably 30to 50 mole-%, more preferably 35 to 45 mole-% of charged sterols areused.
 13. The method according to any of the preceding claims,characterized in that more than 10 mole-%, preferably more than 40mole-%, and more preferably more than 60 mole-% of phospholipids areused.
 14. The method according to any of the preceding claims,characterized in that the lipid membrane is present above its respectivephase transition temperature.
 15. The method according to any of thepreceding claims, characterized in that the template particles arepresent in the form of an oil-in-water emulsion.
 16. The methodaccording to any of the preceding claims, characterized in that theemulsions include active substances in their oil phase.
 17. The methodaccording to any of the preceding claims, characterized in that natural,synthetic polymers, copolymers or block polymers comprised of at leasttwo different monomers and/or mixed forms of these compounds are used aspolyelectrolytes.
 18. The method according to any of the precedingclaims, characterized in that polysaccharides, natural or syntheticproteins, peptides, homo- or heteropolymers of amino acids, copolymers,block polymers and/or those monomers forming the basis of the syntheticpolymers are used as polyelectrolytes.
 19. The method according to anyof the preceding claims, characterized in that alginic acid, chitosan,pectin, hyaluronic acid, polymannuronic acid, polygalacturonic acid,heparin, gum arabic, Indian traganth, xanthan gum, Carragheen, locustbean gum, and the salts of these compounds, as well as carboxylated,aminated, hydrazylated dextrans, starches, levans, inulins, agaroses,polyacrylic acids, polyacrylamides, polyacrylic esters, and otherpolymers of derivatives of acrylic acid, polyvinylpyrrolidones,polyethyleneimines, polystyrenesulfonic acids, polyallylamines, and/orpolyphosphazenes are used as polyelectrolytes.
 20. The method accordingto any of the preceding claims, characterized in that albumin,hemoglobin, myoglobin, antibodies, enzymes, protease, peptidase,oxidoreductase, lipase, esterase, mutase, isomerase, phospholipase,aminotransferase, acylase, lyase, hydrolase, α₂-macroglobulin,fibrinogen, fibronectin, collagen, vitronectin, protein A, protein G,avidin, streptavidin, concanavalin A, and/or wheat germ agglutinin areused as polyelectrolytes.
 21. Structures in the form of nanocapsules,produced according to one or more of claims 1 to
 20. 22. The structuresaccording to claim 21, characterized in that one or more lipid layersare situated in the interior of said structure.
 23. The structuresaccording to claim 22, characterized in that a water-immiscible oilphase is situated in the interior of said structure.
 24. The structuresaccording to any of the preceding claims, characterized in that activesubstances are situated in the interior of said structure.
 25. Thestructures according to any of the preceding claims, characterized inthat the active substances are part of a coat layer.
 26. The structuresaccording to any of the preceding claims, characterized in that theactive substances are catalysts, biocatalysts, pharmaceutical agents,proteins, oligonucleotides, nucleic acids, crystals, and/or sensormolecules.
 27. Use of the structures according to any of claims 21 to 26as containers or vehicles in pharmaceutical formulations.
 28. Use of thestructures according to any of claims 21 to 26 in biochemicaldiagnostics.
 29. Use of the structures according to any of claims 21 to26 in the production of microcrystals, herbicides, pesticides, and/orpigments.
 30. A device for the continuous coating of liposomes withmultiple polyelectrolyte layers, characterized in that a mixer (30-3X)is provided in the main flow of the liposomes effected by a pump (10),downstream of an at tenuator (20) for each separately supplied inflow ofrequired polyelectrolyte (A-X) effected by a pump (11-1X), one timingelement (41-4X) at a time being arranged between the mixers (30-3X), andone attenuator (21-2X) at a time being arranged between the pumps(11-1X) for feeding polyelectrolyte (A-X) and the mixers (30-3X). 31.The device according to claim 30, characterized in that the mixers(3-3X) arranged in the main flow section are connected by means of atube line, and that the volume of the respective tube line between themixer (30 and 31, 31 and 32, 32 and 33, 33 and 34, 34 and 3X,respectively) forms the timing element (41, 42, 43, 44, 45, and 4X,respectively).